923 
III. CULTIVATION. 
METHODS AND MATERIAL. 
Our general plan and technique for the cultivation of amcebe has not 
materially been altered since our first communication on this subject.® 
A few changes in the details have been made, most of which have been 
designed to meet special conditions. These are brought out in the next 
two chapters, in which pure cultures and symbiosis are discussed. 
Up to the present time we have cultivated over two hundred stems of 
amcebe from water, soil, vegetables, fruits, and other extraneous sources : 
from the stools of man and other animals; from the dysenteric ulcers 
examined post-mortem; and from various experimental amcebic abscesses 
in animals. We have also, in cases of ameebic cystitis, grown the para- 
sites from human urine and twice from liver abscesses in man. In the 
latter cases there were both bacteria and amcebe in the abscesses, and, 
in one case, cultures made from the discharges after operation were only 
successful on plates which had previously been inoculated with cultures 
of the bacteria found in the liver abscess. : 
No difficulty, as was mentioned in the first paper, has been experienced 
in cultivating any amceba from water, soil, or other extraneous source. 
However, the problem with those from the human or other animal 
intestine is not always so simple, and in a certain number of such cases 
we have even been entirely unable to grow the parasites. T’he most 
important point to be considered in this connection is that this statement 
is equally true no matter whether the colon infection ts a natural one or 
whether the amebe were originally introduced in the intestine from 
cultures in which they were multiplying profusely. To a certain extent 
at least, this difficulty is brought about by inability to produce satisfactory 
symbiosis ; a further discussion of the causes underlying this phenomenon. 
will be given in the appropriate portion of this paper. 
We have found that, whereas when first isolated the parasites thrive 
best in neutral or slightly alkaline media, this preference can be altered 
by carefully and gradually increasing the acidity, and by this method 
the amoebe eventually can be made to propagate in surroundings in 
which the percentage of acid is greater than it is ever found to be in the 
normal stomach; it is also true that encysted amcbe, after days of 
exposure to quite strong acid solutions, will again develop when they are 
placed upon satisfactory media. 
This renders it evident that vegetables and other substances contami- 
nated with amcebx can not be rendered harmless by treatment with weak 
acids and that the ingestion of acids can not be relied on as a prophylactic 
measure. ‘This statement is rendered the more positive by the fact that 
our experimental work has shown that such measures are of even less 
* Publications of the Bureau of Government Laboratories, Manila (1904), No. 18. 
